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A Simple One-Dimensional Solid-State NMR Method to Characterize the Nuclear Spin Interaction Tensors Associated with the Peptide Bond
JOURNAL OF MAGNETIC RESONANCE ARTICLE NO.
133, 204 –206 (1998)
MN981442
A Simple One-Dimensional Solid-State NMR Method to Characterize the Nuclear Spin Interaction Tensors Associated with the Peptide Bond Dong-Kuk Lee and A. Ramamoorthy1 Biophysics Research Division and Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Received November 6, 1997; revised March 6, 1998
We propose a simple one-dimensional RF pulse sequence for the study of chemical shift and heteronuclear dipolar coupling tensors of oriented as well as unoriented solids. An off-resonance RF decoupling of protons during the signal acquisition of less sensitive nuclei is used to suppress homonuclear 1H–1H dipolar interactions. This method is experimentally demonstrated on peptide samples selectively labeled with 15N isotope. © 1998 Academic Press Key Words: solid-state NMR; tensors; Lee–Goldburg; SLF; peptide bond.
Multidimensional NMR experiments have become important to study the structure and dynamics of molecules in the solid state (1–5). One of the commonly used solid-state NMR methods is the separated-local-field (SLF) experiment (1), yielding two-dimensional correlation of the chemical shift and dipolar interactions. The magnitudes and orientations of spin interaction tensors measured from an SLF experiment provide very powerful information for characterizing rapid, large-amplitude motions in rigid solids and also to interpret the relaxation rates measured through solution NMR spectroscopy (6). Several versions of high resolution two-dimensional SLF experiments (2–5) have been developed based on the use of multiple RF pulse sequences to suppress 1H–1H dipolar couplings. These high-resolution two-dimensional experiments are also routinely used to measure 15N chemical shifts as well as 1 H–15N dipolar couplings of peptides oriented in lipid bilayers in order to study the backbone conformation and dynamics of the peptide (7, 8). In this paper, we present a one-dimensional experiment to study the chemical shift and heteronuclear dipolar coupling tensors from oriented as well as unoriented solid samples. This method is experimentally demonstrated on a model peptide sample selectively labeled with 15N isotope. It may be mentioned here that similar one-dimensional experiments have been used to study the chemical shift and heteronuclear dipolar coupling tensors pertaining to directly bonded heteronuclei not involving protons (9 –18). In the one-dimensional experiment, transverse magnetization of protons prepared with a 90° pulse is cross-polarized to 1
To whom correspondence should be addressed.
1090-7807/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
15
N nuclei using the standard Hartmann–Hahn procedure. After cross-polarization, the 15N magnetization is sampled under an off-resonance irradiation of protons. It is well known that an off-resonance irradiation of protons changes both homonuclear and heteronuclear dipolar interactions (19 –22). On the other hand, an on-resonance decoupling of protons results in a complete suppression of heteronuclear dipolar interaction present in the system. The effect of an off-resonance irradiation on dipolar couplings can be varied by changing either the offset frequency or the RF field strength of the irradiation. At the same time, by proper selection of an off-resonance RF field with an effective field pointing at the magic angle, 1H–1H dipolar coupling can be suppressed to a great extent and 1 H–15N dipolar couplings can be scaled by a factor of 0.58. The time evolution of the observable part of the final density matrix under the one-dimensional pulse sequence can be written as S x 3 cos~0.58 pv ISt$S xcos~ d St! 1 S ysin~ d St!% 1 2 sin~0.58 pv ISt!$S yI zcos~ d St! 1 S xI zsin~ d St!%,
[1]
where I and S represent the 1H and 15N nuclei, respectively, dS is the chemical shift of S spins, and vIS is the I–S dipolar coupling. From Eq. [1], it is clear that two absorbance mode signals result, at frequencies (dS 1 0.58pvIS) and (dS 2 0.58pvIS) radians per second. In the case of a single crystal or an oriented sample, dS depends on the orientation of the sample with respect to the external magnetic field. On the other hand, for powders, dS represents the anisotropic chemical shift, and the expression for dS is
d S 5 s 11Ncos2f sin2u 1 s 22Nsin2f sin2u 1 s 33Ncos2u ,
[2]
where siiN (ii 5 11, 22, 33) are the principal elements of the 15 N chemical shift tensor, u is the angle between the s33N axis and the applied external magnetic field direction, and f is the angle between the projection of the vector representing the external magnetic field direction on the s11N –s22N plane and
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FIG. 1. 15N signal of a 50-mg polycrystalline sample of N-acetylvaline labeled with 15N isotope obtained with (a) no 1H decoupling, (b) on-resonance 1 H decoupling, and (c) irradiation of protons at the magic angle. (d) is the simulated spectrum best fitting with the dipolar-chemical shift spectrum in (c). Spectra (a), (b), and (c) are the result of coadding 20,000, 256, and 2500 transients, respectively, from cross-polarization with 1-ms mix time. A relaxation delay of 3 s was used in all the experiments. Interestingly, a onedimensional dipolar-chemical shift spectrum obtained with even 40 scans is sufficient to characterize the reported spin interaction tensors.
the s11N axis. The dipolar coupling frequency, vIS, under the off-resonance decoupling condition is expressed as
v IS 5
0.58 D IS@1 2 3$sin u sin b cos~ f 2 a ! 2 1 cos u cos b !% 2#,
[3]
where DIS is given as mo\gIgS/4pr3IS, b is the angle between the s33N and the N–H bond vectors, and a is the angle between the s11N axis and the projection of the N–H bond on the s11N–s22N plane (2). u and f are the same as defined in Eq. [2]. Therefore, 15 N signal acquired under off-resonance decoupling of protons will be useful to characterize the 15N chemical shift and 1H– 15 N dipolar coupling tensors in the molecular frame. All of the experiments were performed on a Chemagnetics Infinity 400-MHz spectrometer with a 9.4-T wide-bore JMT 400/89 magnet. A 5-mm Chemagnetics probe double-tuned to 15 N and 1H nuclei resonating at frequencies 40.59 and 400.56 MHz, respectively, were used. An RF field strength of 55.6 kHz was used in both the RF channels. During 15N signal acquisition an offset of 38.5 kHz was used to set the Lee– Goldburg condition. Experiments were performed on a powder sample of a model peptide, N-acetyl-[15N] DL-valine (NAV), labeled with 15N isotope at the amide site. Experimental spectra are given in Fig. 1. 15N chemical shift spectra obtained without any 1H decoupling, with on-resonance 1H decoupling,
205
and with an off-resonance decoupling (19) are given in Figs. 1A, B, and C, respectively. The experimental spectrum in Fig. 1c was simulated to determine the 15N chemical shift and 1 H–15N dipolar coupling tensors in the molecular frame. The best-fitting simulated spectrum is given in Fig. 1d. The simulated spectrum was obtained with the following parameters: s11N 5 56.9 6 0.5, s22N 5 78.3 6 0.5, s33N 5 235.3 6 1 ppm, DIS 5 9.7 6 0.1 kHz, a 5 0°, and b 5 19° 6 2°. Principal elements of the chemical shift tensor were directly measured from Fig. 1B and the spectrum was referenced to NH3 (liquid, 25°C) by setting the observed 15N signal of solid (15NH4)2SO4 to 26.8 ppm. siiN values were confirmed from the isotropic chemical shift frequencies of the 15N MAS spectrum of the NAV powder sample obtained with a 6-kHz spinning frequency. Since there are two magnetically inequivalent molecules in a unit cell of NAV, two isotropic chemical shift peaks were observed at 123.5 and 119 ppm. Error in the tensor values presented in this work due to two different 15N resonances is negligible as the signal intensity of the high-field resonance peak is about 8% of the low-field resonance peak. The best-fitting simulated spectrum shown in Fig. 1D was obtained by comparing the intensity ratios of the shoulders as well as their frequency separations with the experimental spectrum given in Fig. 1C. The intensity ratio of the shoulders of the powder pattern is highly sensitive to the change in b value. The spectrum is somewhat less sensitive to the a angle when the b is a small value; however, it is possible to estimate a to be in the range of 0–10°. The frequency separation of the shoulders is sensitive to the 1H–15N dipolar coupling and the widths of the low-frequency shoulders are sensitive to a angle. As a result, a change of 1° for b, 5° for a, and 100 Hz for the 1H–15N dipolar coupling is transparent from the one-dimensional dipolar-chemical shift spectrum. In the simulations, we assumed that the 1H–15N dipolar coupling is collinear with the N–H bond. Therefore, the least shielded component of the 15N chemical shift tensor, s33N, is 19° away from the direction of the N–H bond. This value agrees well with other reported studies in the literature (11–13, 23–26). We have also analyzed the performance of this method on several peptide samples, both single-crystalline and polycrystalline, selectively labeled with 15N isotope. In the case of a singlecrystal sample, DANTE-90° was used before the signal acquisition to select an 15N resonance in order to measure the dipolar coupling directly from the resultant Pake doublet shifted by the 15 N chemical shift. The measured 15N chemical shift and 1H–15N dipolar coupling could be used to determine the orientation of the peptide plane relative to the external magnetic field of the spectrometer (7, 8). Linewidth measured from the dipolar-chemical shift spectrum of a single crystal was on the order of a kilohertz. This is not as narrow as the dipolar spectral lines obtained through the PISEMA experiment (4). Nevertheless, these line widths are comparable with most of the high-resolution two-dimensional SLF methods (4). There are several advantages in using this method to char-
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acterize nuclear spin interaction tensors. Since this is a onedimensional experiment, resolution of the dipolar spectral lines depends on the acquisition time and the number of points sampled during the acquisition of the 15N signal. On the other hand, in a two-dimensional SLF experiment, it depends on the number of points sampled in the indirect frequency, or heteronuclear dipolar, dimension. Therefore, determination of tensors using this method is faster and easier to implement compared to the traditional 2D SLF experiments. The one-dimensional dipolar-chemical shift spectrum obtained using this method is simple to interpret and to measure for tensor values. This method would be highly useful in studying biological solids labeled with specific isotopes, for example, 15N or 13C, as there exists a common difficulty in obtaining sizable amount of such samples for NMR experiments (27). For example, 15N chemical shift and 1H–15N dipolar coupling pertaining to an amide site of a membrane-associated peptide or protein labeled with 15 N at a specific site, oriented in phospholipid bilayers either mechanically or magnetically, could be measured in order to determine its backbone conformation. ACKNOWLEDGMENTS This research was supported by the research funds from the Department of Chemistry and Biophysics Research Division at the University of Michigan.
REFERENCES 1. J. S. Waugh, Uncoupling of local field spectra in nuclear magnetic resonance: Determination of atomic positions in solids, Proc. Natl. Acad. Sci. USA 73, 1394 –1397 (1976). 2. M. Linder, A. Hohener, and R. R. Ernst, Orientation of tensorial interactions determined from two-dimensional NMR powder spectra, J. Chem. Phys. 73, 4959 – 4970 (1980). 3. M. G. Munowitz, R. G. Griffin, G. Bodenhausen, and T. H. Huang, Two-dimensional rotational spin-echo nuclear magnetic resonance in solids: Correlation of chemical shift and dipolar interactions. J. Am. Chem. Soc. 103, 2529 –2533 (1981). 4. C. H. Wu, A. Ramamoorthy, and S. J. Opella, High-resolution heteronuclear dipolar solid-state NMR spectroscopy. J. Magn. Reson. A 109, 270 –272 (1994). 5. A. Ramamoorthy, C. H. Wu, and S. J. Opella, Three-dimensional solid-state NMR experiment that correlates the chemical shift and dipolar coupling frequencies of two heteronuclei, J. Magn. Reson. B 107, 88 –90 (1995). 6. G. Wagner, S. Hyberts, and J. W. Peng, Study of protein dynamics by NMR, in ‘‘NMR of Proteins’’ (G. M. Clore and A. M. Gronenborn, Eds.), pp. 220 –257, Macmillan, London (1993). 7. A. Ramamoorthy, F. M. Marassi, M. Zasloff, and S. J. Opella, Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers, J. Biomol. NMR 6, 329 –334 (1995). 8. R. Jelinek, A. Ramamoorthy, and S. J. Opella, High-resolution three-dimensional solid-state NMR spectroscopy of a uniformly 15 N-labeled protein, J. Am. Chem. Soc. 117, 12348 –12349 (1995). 9. K. G. Valentine, A. L. Rockwell, L. M. Gierasch, and S. J. Opella, 15N chemical-shift tensor of the imide nitrogen in the alanyl–prolyl peptide bond, J. Magn. Reson. 73, 519 –523 (1987). 10. T. G. Oas, C. J. Hartzell, T. J. McMahon, G. P. Drobny, and F. W. Dahlquist, The carbonyl 13C chemical shift tensors of five peptides
determined from 15N dipole-coupled chemical shift powder patterns, J. Am. Chem. Soc. 109, 5956 –5962 (1987). 11. T. G. Oas, C. J. Hartzell, F. W. Dahlquist, and G. P. Drobny, The amide 15N chemical shift tensors of four peptides determined from 13 C dipolar-coupled chemical shift powder patterns, J. Am. Chem. Soc. 109, 5962–5966 (1987). 12. C. J. Hartzell, M. Whitfield, T. G. Oas, and G. P. Drobny, Determination of the 15N and 13C chemical shift tensors of L-[13C]Alanyl-L[15N]alanine from the dipole-coupled powder patterns, J. Am. Chem. Soc. 109, 5966 –5969 (1987). 13. Q. Teng and T. A. Cross, The in situ determination of the 15N chemical shift tensor orientation in a Polypeptide, J. Magn. Reson. 85, 439 – 447 (1989). 14. R. E. Wasylishen, G. H. Penner, W. P. Power, and R. D. Curtis, Dipolar NMR-spectra of the oxime moiety in (e)-acetophenone oxime— carbon and nitrogen chemical shielding anisotropies, J. Chem. Soc. 111, 6082– 6086 (1989). 15. F. Separovic, R. Smith, C. S. Yannoni, and B. A. Cornell, Molecular sequence effect on the 13C carbonyl chemical shift shielding tensor, J. Am. Chem. Soc. 112, 8324 – 8328 (1990). 16. R. D. Curtis, G. H. Penner, W. P. Power, and R. E. Wasylishen, Dipolar-chemical shift NMR-spectra of the carbon nitrogen linkage in benzylideneaniline— carbon and nitrogen chemical shielding anisotropies, J. Phys. Chem. 94, 4000 – 4006 (1990). 17. D. A. Burwell, K. G. Valentine, and M. E. Thompson, 31P and 13C chemical-shift tensors in zirconium phosphonates, J. Magn. Reson. 97, 498 –510 (1992). 18. M. D. Lumsden, R. E. Wasylishen, K. Eichele, M. Schindler, G. H. Penner, W. P. Power, and R. D. Curtis, Carbonyl carbon and nitrogen chemical shift tensors of the amide fragment of acetanilide and N-methylacetanilide, J. Am. Chem. Soc. 116, 1403–1413 (1994). 19. M. Lee and W. I. Goldburg, Nuclear-magnetic-resonance line narrowing by a rotating rf field, Phys. Rev. 140A, 1261–1271 (1965). 20. M. Mehring and J. S. Waugh, Magic-angle NMR experiments in solids, Phys. Rev. B5, 3459 –3471 (1972). 21. A. Bielecki, A. C. Kolbert, H. J. M. de Groot, R. G. Griffin, and M. H. Levitt, Frequency-switched Lee–Goldburg sequences in solids, Adv. Magn. Reson. 14, 111–124 (1990). 22. R. K. Hester, J. L. Ackerman, B. L. Neff, and J. S. Waugh, Separated local field spectra in NMR: Determination of structure of solids, Phys. Rev. Lett. 36, 1081–1083 (1976). 23. C. H. Wu, A. Ramamoorthy, L. M. Gierasch, and S. J. Opella, Simultaneous characterization of the amide 1H chemical shift, 1H– 15 N dipolar, and 15N chemical shift interaction tensors in a peptide bond by three-dimensional solid-state NMR Spectroscopy, J. Am. Chem. Soc. 117, 6148 – 6149 (1995). 24. G. S. Harbison, L. W. Jelinski, R. E. Stark, D. A. Torchia, J. Herzfeld, and R. G. Griffin, 15N chemical shift and 15N–13C dipolar tensors for the peptide bond in [1-13C]glycyl[15N]glycine hydrochloride monohydrate, J. Magn. Reson. 60, 79 – 82 (1984). 25. Y. Hiyama, C.-H. Niu, J. V. Silverton, A. Bavoso, and D. A. Torchia, Determination of 15N chemical shift tensor via 15N–2H dipolar coupling in boc-glycylglycyl[15N]glycine benzyl ester, J. Am. Chem. Soc. 110, 2378 –2383 (1988). 26. W. Mai, W. Hu, C. Wang, and T. A. Cross, Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: The polypeptide backbone of gramicidine A in lipid bilayers, Protein Sci. 2, 532–542 (1993). 27. T. A. Cross, and S. J. Opella, Solid-state NMR structural studies of peptides and proteins in membranes, Curr. Opin. Struct. Biol. 4, 574 –581 (1994).
133, 204 –206 (1998)
MN981442
A Simple One-Dimensional Solid-State NMR Method to Characterize the Nuclear Spin Interaction Tensors Associated with the Peptide Bond Dong-Kuk Lee and A. Ramamoorthy1 Biophysics Research Division and Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Received November 6, 1997; revised March 6, 1998
We propose a simple one-dimensional RF pulse sequence for the study of chemical shift and heteronuclear dipolar coupling tensors of oriented as well as unoriented solids. An off-resonance RF decoupling of protons during the signal acquisition of less sensitive nuclei is used to suppress homonuclear 1H–1H dipolar interactions. This method is experimentally demonstrated on peptide samples selectively labeled with 15N isotope. © 1998 Academic Press Key Words: solid-state NMR; tensors; Lee–Goldburg; SLF; peptide bond.
Multidimensional NMR experiments have become important to study the structure and dynamics of molecules in the solid state (1–5). One of the commonly used solid-state NMR methods is the separated-local-field (SLF) experiment (1), yielding two-dimensional correlation of the chemical shift and dipolar interactions. The magnitudes and orientations of spin interaction tensors measured from an SLF experiment provide very powerful information for characterizing rapid, large-amplitude motions in rigid solids and also to interpret the relaxation rates measured through solution NMR spectroscopy (6). Several versions of high resolution two-dimensional SLF experiments (2–5) have been developed based on the use of multiple RF pulse sequences to suppress 1H–1H dipolar couplings. These high-resolution two-dimensional experiments are also routinely used to measure 15N chemical shifts as well as 1 H–15N dipolar couplings of peptides oriented in lipid bilayers in order to study the backbone conformation and dynamics of the peptide (7, 8). In this paper, we present a one-dimensional experiment to study the chemical shift and heteronuclear dipolar coupling tensors from oriented as well as unoriented solid samples. This method is experimentally demonstrated on a model peptide sample selectively labeled with 15N isotope. It may be mentioned here that similar one-dimensional experiments have been used to study the chemical shift and heteronuclear dipolar coupling tensors pertaining to directly bonded heteronuclei not involving protons (9 –18). In the one-dimensional experiment, transverse magnetization of protons prepared with a 90° pulse is cross-polarized to 1
To whom correspondence should be addressed.
1090-7807/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
15
N nuclei using the standard Hartmann–Hahn procedure. After cross-polarization, the 15N magnetization is sampled under an off-resonance irradiation of protons. It is well known that an off-resonance irradiation of protons changes both homonuclear and heteronuclear dipolar interactions (19 –22). On the other hand, an on-resonance decoupling of protons results in a complete suppression of heteronuclear dipolar interaction present in the system. The effect of an off-resonance irradiation on dipolar couplings can be varied by changing either the offset frequency or the RF field strength of the irradiation. At the same time, by proper selection of an off-resonance RF field with an effective field pointing at the magic angle, 1H–1H dipolar coupling can be suppressed to a great extent and 1 H–15N dipolar couplings can be scaled by a factor of 0.58. The time evolution of the observable part of the final density matrix under the one-dimensional pulse sequence can be written as S x 3 cos~0.58 pv ISt$S xcos~ d St! 1 S ysin~ d St!% 1 2 sin~0.58 pv ISt!$S yI zcos~ d St! 1 S xI zsin~ d St!%,
[1]
where I and S represent the 1H and 15N nuclei, respectively, dS is the chemical shift of S spins, and vIS is the I–S dipolar coupling. From Eq. [1], it is clear that two absorbance mode signals result, at frequencies (dS 1 0.58pvIS) and (dS 2 0.58pvIS) radians per second. In the case of a single crystal or an oriented sample, dS depends on the orientation of the sample with respect to the external magnetic field. On the other hand, for powders, dS represents the anisotropic chemical shift, and the expression for dS is
d S 5 s 11Ncos2f sin2u 1 s 22Nsin2f sin2u 1 s 33Ncos2u ,
[2]
where siiN (ii 5 11, 22, 33) are the principal elements of the 15 N chemical shift tensor, u is the angle between the s33N axis and the applied external magnetic field direction, and f is the angle between the projection of the vector representing the external magnetic field direction on the s11N –s22N plane and
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FIG. 1. 15N signal of a 50-mg polycrystalline sample of N-acetylvaline labeled with 15N isotope obtained with (a) no 1H decoupling, (b) on-resonance 1 H decoupling, and (c) irradiation of protons at the magic angle. (d) is the simulated spectrum best fitting with the dipolar-chemical shift spectrum in (c). Spectra (a), (b), and (c) are the result of coadding 20,000, 256, and 2500 transients, respectively, from cross-polarization with 1-ms mix time. A relaxation delay of 3 s was used in all the experiments. Interestingly, a onedimensional dipolar-chemical shift spectrum obtained with even 40 scans is sufficient to characterize the reported spin interaction tensors.
the s11N axis. The dipolar coupling frequency, vIS, under the off-resonance decoupling condition is expressed as
v IS 5
0.58 D IS@1 2 3$sin u sin b cos~ f 2 a ! 2 1 cos u cos b !% 2#,
[3]
where DIS is given as mo\gIgS/4pr3IS, b is the angle between the s33N and the N–H bond vectors, and a is the angle between the s11N axis and the projection of the N–H bond on the s11N–s22N plane (2). u and f are the same as defined in Eq. [2]. Therefore, 15 N signal acquired under off-resonance decoupling of protons will be useful to characterize the 15N chemical shift and 1H– 15 N dipolar coupling tensors in the molecular frame. All of the experiments were performed on a Chemagnetics Infinity 400-MHz spectrometer with a 9.4-T wide-bore JMT 400/89 magnet. A 5-mm Chemagnetics probe double-tuned to 15 N and 1H nuclei resonating at frequencies 40.59 and 400.56 MHz, respectively, were used. An RF field strength of 55.6 kHz was used in both the RF channels. During 15N signal acquisition an offset of 38.5 kHz was used to set the Lee– Goldburg condition. Experiments were performed on a powder sample of a model peptide, N-acetyl-[15N] DL-valine (NAV), labeled with 15N isotope at the amide site. Experimental spectra are given in Fig. 1. 15N chemical shift spectra obtained without any 1H decoupling, with on-resonance 1H decoupling,
205
and with an off-resonance decoupling (19) are given in Figs. 1A, B, and C, respectively. The experimental spectrum in Fig. 1c was simulated to determine the 15N chemical shift and 1 H–15N dipolar coupling tensors in the molecular frame. The best-fitting simulated spectrum is given in Fig. 1d. The simulated spectrum was obtained with the following parameters: s11N 5 56.9 6 0.5, s22N 5 78.3 6 0.5, s33N 5 235.3 6 1 ppm, DIS 5 9.7 6 0.1 kHz, a 5 0°, and b 5 19° 6 2°. Principal elements of the chemical shift tensor were directly measured from Fig. 1B and the spectrum was referenced to NH3 (liquid, 25°C) by setting the observed 15N signal of solid (15NH4)2SO4 to 26.8 ppm. siiN values were confirmed from the isotropic chemical shift frequencies of the 15N MAS spectrum of the NAV powder sample obtained with a 6-kHz spinning frequency. Since there are two magnetically inequivalent molecules in a unit cell of NAV, two isotropic chemical shift peaks were observed at 123.5 and 119 ppm. Error in the tensor values presented in this work due to two different 15N resonances is negligible as the signal intensity of the high-field resonance peak is about 8% of the low-field resonance peak. The best-fitting simulated spectrum shown in Fig. 1D was obtained by comparing the intensity ratios of the shoulders as well as their frequency separations with the experimental spectrum given in Fig. 1C. The intensity ratio of the shoulders of the powder pattern is highly sensitive to the change in b value. The spectrum is somewhat less sensitive to the a angle when the b is a small value; however, it is possible to estimate a to be in the range of 0–10°. The frequency separation of the shoulders is sensitive to the 1H–15N dipolar coupling and the widths of the low-frequency shoulders are sensitive to a angle. As a result, a change of 1° for b, 5° for a, and 100 Hz for the 1H–15N dipolar coupling is transparent from the one-dimensional dipolar-chemical shift spectrum. In the simulations, we assumed that the 1H–15N dipolar coupling is collinear with the N–H bond. Therefore, the least shielded component of the 15N chemical shift tensor, s33N, is 19° away from the direction of the N–H bond. This value agrees well with other reported studies in the literature (11–13, 23–26). We have also analyzed the performance of this method on several peptide samples, both single-crystalline and polycrystalline, selectively labeled with 15N isotope. In the case of a singlecrystal sample, DANTE-90° was used before the signal acquisition to select an 15N resonance in order to measure the dipolar coupling directly from the resultant Pake doublet shifted by the 15 N chemical shift. The measured 15N chemical shift and 1H–15N dipolar coupling could be used to determine the orientation of the peptide plane relative to the external magnetic field of the spectrometer (7, 8). Linewidth measured from the dipolar-chemical shift spectrum of a single crystal was on the order of a kilohertz. This is not as narrow as the dipolar spectral lines obtained through the PISEMA experiment (4). Nevertheless, these line widths are comparable with most of the high-resolution two-dimensional SLF methods (4). There are several advantages in using this method to char-
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acterize nuclear spin interaction tensors. Since this is a onedimensional experiment, resolution of the dipolar spectral lines depends on the acquisition time and the number of points sampled during the acquisition of the 15N signal. On the other hand, in a two-dimensional SLF experiment, it depends on the number of points sampled in the indirect frequency, or heteronuclear dipolar, dimension. Therefore, determination of tensors using this method is faster and easier to implement compared to the traditional 2D SLF experiments. The one-dimensional dipolar-chemical shift spectrum obtained using this method is simple to interpret and to measure for tensor values. This method would be highly useful in studying biological solids labeled with specific isotopes, for example, 15N or 13C, as there exists a common difficulty in obtaining sizable amount of such samples for NMR experiments (27). For example, 15N chemical shift and 1H–15N dipolar coupling pertaining to an amide site of a membrane-associated peptide or protein labeled with 15 N at a specific site, oriented in phospholipid bilayers either mechanically or magnetically, could be measured in order to determine its backbone conformation. ACKNOWLEDGMENTS This research was supported by the research funds from the Department of Chemistry and Biophysics Research Division at the University of Michigan.
REFERENCES 1. J. S. Waugh, Uncoupling of local field spectra in nuclear magnetic resonance: Determination of atomic positions in solids, Proc. Natl. Acad. Sci. USA 73, 1394 –1397 (1976). 2. M. Linder, A. Hohener, and R. R. Ernst, Orientation of tensorial interactions determined from two-dimensional NMR powder spectra, J. Chem. Phys. 73, 4959 – 4970 (1980). 3. M. G. Munowitz, R. G. Griffin, G. Bodenhausen, and T. H. Huang, Two-dimensional rotational spin-echo nuclear magnetic resonance in solids: Correlation of chemical shift and dipolar interactions. J. Am. Chem. Soc. 103, 2529 –2533 (1981). 4. C. H. Wu, A. Ramamoorthy, and S. J. Opella, High-resolution heteronuclear dipolar solid-state NMR spectroscopy. J. Magn. Reson. A 109, 270 –272 (1994). 5. A. Ramamoorthy, C. H. Wu, and S. J. Opella, Three-dimensional solid-state NMR experiment that correlates the chemical shift and dipolar coupling frequencies of two heteronuclei, J. Magn. Reson. B 107, 88 –90 (1995). 6. G. Wagner, S. Hyberts, and J. W. Peng, Study of protein dynamics by NMR, in ‘‘NMR of Proteins’’ (G. M. Clore and A. M. Gronenborn, Eds.), pp. 220 –257, Macmillan, London (1993). 7. A. Ramamoorthy, F. M. Marassi, M. Zasloff, and S. J. Opella, Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers, J. Biomol. NMR 6, 329 –334 (1995). 8. R. Jelinek, A. Ramamoorthy, and S. J. Opella, High-resolution three-dimensional solid-state NMR spectroscopy of a uniformly 15 N-labeled protein, J. Am. Chem. Soc. 117, 12348 –12349 (1995). 9. K. G. Valentine, A. L. Rockwell, L. M. Gierasch, and S. J. Opella, 15N chemical-shift tensor of the imide nitrogen in the alanyl–prolyl peptide bond, J. Magn. Reson. 73, 519 –523 (1987). 10. T. G. Oas, C. J. Hartzell, T. J. McMahon, G. P. Drobny, and F. W. Dahlquist, The carbonyl 13C chemical shift tensors of five peptides
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